Recombinant proteins are extensively used in a variety of applications and therefore the demand for their efficient production is high. Recombinant proteins and polypeptides can be produced in heterologous host cells, with bacterial cells such as Escherichia coli (E. coli) being the most widely used hosts for recombinant protein production both for research and pharmaceutical applications. In particular, extreme E. coli cell robustness, fast and simple cultivation, easy genetic manipulation, enormous amounts of available physiological data and molecular biology tools are the reasons that make this microbe so widespread for recombinant protein production applications.
Despite these positive features, most heterologous protein production attempts in bacterial cells are ending with significantly reduced functional protein yields due target protein aggregation and/or improper folding. Many examples show that in general bacterial cell protein folding machinery is not adapted for a high level accumulation of “foreign” protein molecules with unusual features for the host: disulfide bonds, high hydrophobicity, etc.
Aggregation of heterologous protein in E. coli production hosts is a common phenomenon; a consequence of the inability of the host's folding machinery to cope with the rapidly accumulating target protein folding and/or to facilitate efficient stabilization of SH groups, or contribute to the formation and/or reorganization of correct disulfide bonds (see recent reviews Baneyx F et al. 2004 [1], Francis D M et. al 2010 [2]).
So far, the main research focus in this field is directed towards issues relating to correct disulfide bond formation, since these bonds are often important structural features of eukaryotic proteins. Direction of expressed recombinant protein into the cytoplasmic space of E. coli strains carrying trx/gor mutations or to the naturally oxidative periplasmic compartment are the most common approaches for improvement of heterologous protein disulfide bond formation (de Marco A et al. 2009 [3]). The efficiency of disulfide bond formation, or so-called disulfide shuffling, in E. coli periplasmic space is usually achieved by using in cultivation medium low molecular weight additives, which modify SH groups thereby aiding protein folding. For example, reduced/oxidized glutathione (GSH/GSSG) and arginine can easily penetrate the outer membrane and effect disulfide bond formation of recombinant proteins, propagation of which is directed to the periplasmic space. The utilization of low molecular weight agents GSH/GSSG for enhancement of disulfide bond formation in the periplasmic space for facilitated protein folding in respect of model proteins expressed and efficiently transported to the periplasmic space in a soluble state was disclosed by Wunderlich M et. al 1993 [4] and Walker K W et. al 1994 [5] (see also EP0510658B1).
Analogous, but more sophisticated in vivo folding/disulfide bond formation approaches for periplasmic space are based on the utilization of co-secreted prokaryotic disulfide oxidoreductases DsbA or DsbC as described in U.S. Pat. No. 5,639,635 and by Wunderlich M et al. 1993 [4], or eukaryotic protein disulfide isomerase (PDI) (Ostermeier M et al. 1996 [6]). Another example is where the presence of co-secreted cytoplasmic chaperones DnaJ or Hsp25 in combination with low molecular medium additives, such as L-arginine and 5 mM reduced GSH, facilitated folding of disulfide bonds containing proteins: a truncated version of tissue-type plasminogen activator, proinsulin and a single-chain antibody fragment, in the periplasmic space of E. coli BL21 (DE3) Schäffner J et al. 2001 [7].
Externally added components can also affect the redox state of disulfide bonds containing proteins in cytoplasm, as it was reported by Gill et al. 1998 [8]. Folding and activity of chloramphenicol acetyltransferase (CAT) in the cytoplasmic compartment was altered due to the presence of dithiothreitol (DTT) in the cultivation medium and resulted in enzyme inactivation but the mechanism underlying these changes is not clear. Further, the presence of DTT was indicated to have a negative effect on cellular physiology, causing in particular the elevated synthesis of host proteases.
There are several reports describing negative effect of reducing agent capable of diffusion through cell membrane, DTT, on disulfide bond formation/bond disruption in eukaryotic yeast and mammalian cells. The DTT when added to the cultivation medium, prevented disulfide bond formation of several proteins in ER and cytoplasmic space of living yeast cells thereby rendering those protein inactive. For example, Braakman and co-workers have demonstrated disruption of disulfide bond formation of influenza hemagglutinin (HAO) and induction of reduction of already oxidized HAO in endoplasmic reticulum (ER) (Braakman I et al. 1992 [9]) by addition of DTT to the culture medium. Paunola and coworkers used DTT to confirm lack of disulfide bond formation during the folding of beta-lactamase (Paunola et al. 1998 [10]), as the addition of this reagent had no effect on enzyme activity. However, their work has also led scientists to believe that addition of DTT into cultivation medium has no effect on folding/activity of the proteins that do not contain disulfide bonds in their structure.
Another yeast cell related example was reported by Jämsä E and co-workers, where DTT-mediated disulfide bond disruption resulted in retention in the ER of vacuolar enzyme carboxypeptidase Y (CPY) and of secretory stress-protein hsp150. The proteins were reoxidized after DTT was washed out from the cells (Jämsä E et al. 1994 [11]).
The effects of DTT on disulfide bond formation in cytoplasmic space of mammalian cells were investigated by Valetti C et al. 1994 [12]. Mezghrani A and coworkers used DTT for manipulation of PDI redox state in experiments demonstrating oxidative folding capabilities of human Ero1-Lalpha and Ero1-Lbeta (hEROs) in living HeLa mammalian cells (Mezghrani A et al. 2001 [13]).
Raines discloses the dithiol catalysis approach, which aims to facilitate disulfide bond formation of heterologous protein in living cells (in vivo) and in vitro (U.S. Pat. No. 5,910,435). The method is based on utilization of organic dithiol molecules N,N′-bis(2-mercaptoacetyl)-1,2-diaminocyclohexane with specifically defined chemical properties: pKa more than about 8.0 and a standard reduction potential of greater than about −0.25 volts. Such organic dithiol molecules were useful in catalysis of correct disulfide bond formation in the target protein in living yeast cells, which were deficient in protein disulfide isomerase (PDI) expression. The ability of organic dithiol molecules to catalyze disulfide bond formation in vitro was demonstrated on disulfide bond containing ribonuclease A.
However, few prior art documents address the issue of improving the production of proteins, such as ribonuclease inhibitor (RI), which contain a number of reduced cysteines that are vital to the function of the protein.
The members of the ribonuclease inhibitor group represent a specific subfamily within the large group of proteins with a very special protein fold—the Leucine Rich Repeat (LRR) proteins (Kobe B et. al 2001 [14]). LRR proteins share very interesting features, which make them a unique group of proteins. RI has an unusual non-globular flexible horseshoe like structure, which is highly conserved between different species. The core of ribonuclease inhibitor molecule is composed of hydrophobic 15-16 LRR motifs. Each of the LRR consists of a structural unit of 28 to 29 amino acids forming an α-helix and β-strand connected by loops (Dickson K A et al 2005 [15]). RI has a very high leucine content (18%), but also contains 30-32 cysteine residues (6.5-7%). In contrast to other LRR motif containing proteins where the cysteines are involved in maintaining protein structure (see e.g. Park H et. al. 2008 [16]), all cysteines in RI are reduced, a feature that is very important for its activity, i.e. substrate interaction. Oxidation of free SH groups in RI is highly cooperative and leads to enzyme inactivation and even denaturation (Fominaya J M et al. 1992 [17], Dickson K A et al. 2005 [15]).
Production of RI has been a challenge due to its flexible structure, amino acid repeats and reduced cysteines. In particular, the protein strongly aggregates if expressed in E. coli, even when folding takes place in the cytoplasm of bacteria, which is known to provide a reducing environment that prevents disulphide bond formation. Further, so far reported RI production attempts in yeast Saccharomyces cerevisiae (Vicentini A M et. al 1990 [18]) and in E. coli (Lee F S et al. 1989 [19], Klink TA, 2001 [20]) ended up with a low overall yield either due to low production levels and/or high RI insolubility. Recently, high amounts of soluble RI were produced in E. coli cells after the protein the MBP tag (≈32 kDa) was fused to the N-terminus. These results suggest that the addition of a tag may provide a solution to the problem of protein aggregation during production (Siurkus J et al. 2010 [21]).
Despite the existing body of research on improved bioproduction processes for disulfide bond forming heterologous proteins in E. coli, little is known about ways to improve yields of properly folded target recombinant proteins that possess reduced cysteines and, hence, their folding is not dependent on disulfide bond formation, or alternative ways to address the problem of insoluble protein accumulation. Therefore there is constant need in the industry for improved, more efficient methods for production of these recombinant proteins in host cell systems.